Tumor Exosome-Mediated MDSC Activation

Abstract

To the Editor-in-Chief: In a recent issue of The American Journal of Pathology, Xiang et al1 published their results concerning the effects of tumor-derived exosomes on myeloid-derived suppressor cell (MDSC) biology. They compared the biological effects of exosomes derived from in vitro cultured B16 tumor cells (termed C-exo for culture exosome) and exosomes derived from in vivo grown B16 tumor (termed P-exo for primary exosomes). They reported that P-exo induce Toll-like receptor 2 (TLR2)–independent MDSC activation and expansion, whereas C-exo activate and expand MDSC in a TLR2-dependent manner. These data are in contrast with our own recent article demonstrating MDSC activation through TLR2 ligation by heat shock protein 72 (Hsp72) expressed on exosomes from CT26 tumors.2 In their discussion, Xiang et al1 propose a hypothesis to explain this discrepancy between C-exo and P-exo. Here, we provide our point of view about this difference. Xiang et al1 propose that P-exo harbor a TLR2-independent mechanism of MDSC activation that is different from C-exo. This is probably true. Indeed, in another study from the same group, Xiang et al3 provided evidence that TS/A tumor cell lines produce exosomes containing prostaglandin E2 (PGE2), and it has previously been shown that PGE2 alone is sufficient to induce MDSC expansion and activation in a TLR2-independent manner.4 However, in the models used in our study, we provide evidence that there is no detectable PGE2 in tumor-derived exosomes. Xiang et al1 assert that our work demonstrated that “tumor exosomes trigger MDSC expansion via activation of STAT3.” However, this assumption is false. In our model, we demonstrated that there are indeed two distinct signals: tumor-derived exosomes account for MDSC activation (STAT3 phosphorylation and interleukin 6 secretion), whereas tumor-derived soluble factors [namely, granulocyte-macrophage colony-stimulating factor (GM-CSF)] are responsible for expansion. In the EL4 model, we observed that tumors growing in TLR2−/− mice induced MDSC expansion but not MDSC activation compared with wild-type mice. Thus, we can exclude the in vitro effect hypothesized by Xiang et al,1 because, in this in vivo setting, we observed proliferation dissociated from activation. In the model by Xiang et al, for unknown reasons, C-exo induced both STAT3 activation and expansion of MDSC; however, we never observed proliferation of MDSC cultured with exosomes alone. Moreover, Xiang et al already reported that the expansion and proliferation of MDSC in their model may be related to the presence of PGE2. Indeed, we also observed that PGE2 alone could trigger MDSC expansion and proliferation, so we could postulate that exosomes may have contained PGE2 in the model of Xiang et al, whereas we could not detect PGE2 in exosomes from our own models. These data may explain the discrepancy between our works. Furthermore, the preparation of the P-exo raises some methodological concerns. They are prepared from isolated in vivo grown tumors, with less than 5 in vitro passages. Many nontumoral cells, such as myeloid cells or fibroblasts, might be present in the preparation. These cell contaminants could be responsible for the production of PGE2-containing exosomes because it is well known that tumor-infiltrating macrophages may produce PGE25 and exosomes.6 Xiang et al1 also propose that the difference between C-exo and P-exo is based on the passage number of in vitro cultured cells. We work with cell lines obtained from the American Type Culture Collection and used at passage numbers less than 15 to 20, so we assume the derivation from the parental cell line is minimal. Xiang et al also proposed that mycoplasma contamination could be responsible for the TLR2-dependent activation signal, which would be eliminated by the in vivo passage of the tumor. In our study, we routinely tested for mycoplasma contamination, but Xiang et al suspect that the available tests are insufficient. However, in our study, we demonstrated that exosomes derived from mycoplasma-free Hsp72-silenced CT26 were unable to activate MDSC, whereas mycoplasma-free mock CT26-derived exosomes activated MDSC. Further, in vivo Hsp72-silenced tumors were unable to induce STAT3 activation of MDSC, thus supporting the premise that the Hsp72-TLR2-MyD88 pathway is vital in our model. In our opinion, this clearly excludes concerns about a potential effect of mycoplasma contamination because the Hsp72 status alone would not be a determinant otherwise. In summary, we propose that the discrepancy identified by Xiang et al1 between C-exo and P-exo may actually rely on the presence of tumor exosome–associated PGE2 produced by tumor or contaminant cells, which bypass the Hsp72-TLR2-MyD88 signal.